Abstract: Fucoidan is a marine polysaccharide with diverse physiological activities. Endo-1,3-fucoidanase is a favorable tool for the preparation of fucooligosaccharides. Nevertheless, the lack of food-grade fucoidanase with clear cleavage sites has hindered its application. This study aimed to obtain a food-grade endo-1,3-fucoidanase from GH174 family (Fun174Sb) and to investigate its cleavage site. The study demonstrated that the food-grade expression of Fun174Sb could be achieved through the Lactobacillus NICE system. Fun174Sb exhibits significant endo-acting hydrolysis activity towards fucoidan from Holothuria tubulosa (Ht-FUC), with an enzymatic activity of 1.90 U/mL. Fun174Sb showed significant activity within a temperature range of 35-50 °C and a pH range of 7.5-8.5, and it possessed good temperature and pH stability. Furthermore, the structure of the dominant oligosaccharide in the enzymatic product was analyzed using ultra performance size exclusion chromatography-mass spectrum (UPSEC-MS) and nuclear magnetic resonance (NMR). It could be inferred that Fun174Sb cleaves the α-1,3-glycosidic bond between Fucp2 (OSO₃⁻) and Fucp2,4 (OSO₃⁻) in Ht-FUC. The study showed that Fun174Sb expressed in Lactobacillus exhibited favorable biochemical properties and a clear mode of action. It laid the foundation for the application of food-grade fucoidanase.